Lyme disease is the most common vector-borne disease in the United States and is caused by the tick-borne spirochete Borrelia burgdorferi. Maintenance of B. burgdorferi in the environment depends upon a complex enzootic cycle involving a variety of mammalian and avian reservoirs. B. burgdorferi establishes long-term infections in both its tick vector and reservoir hosts, and we are broadly interested in the mechanisms by which B. burgdorferi evades host immune responses and adapts to the very different environments it encounters. In addition to factors such as temperature, pH, metals, and cell density, oxidative stress likely represents an environmental cue and selective pressure for B. burgdorferi. A handful of genes important for conferring resistance to oxidative stress have been identified in B. burgdorferi, primarily based on homology with characterized genes in other organisms. B. burgdorferi appears to have a smaller repertoire of oxidative stress and redox-responsive genes compared to well-studied model organisms such as E. coli, and the targets of oxidative and nitrosative stress also differ between these organisms, suggesting that novel gene functions and pathways may contribute to the oxidative stress response in B. burgdorferi. In the current proposal, we will use Tn-seq to conduct an unbiased, whole-genome search for genes involved in the B. burgdorferi oxidative stress response. Tn-seq relies on high-throughput sequencing technology to quantify the frequency of every mutant in a transposon library before and after a selective pressure. We will expose a B. burgdorferi transposon library to hydrogen peroxide, t-butyl hydroperoxide, and nitric oxide and will confirm the genes identified in the screen by generating targeted deletion and complementation strains. We will then determine the significance of these genes during B. burgdorferi infection of its natural mouse and tick hosts. We will determine the ability of the mutant and complemented B. burgdorferi strains to infect wild-type mice, as well as mice that are unable to produce superoxide, hypochlorous acid, or nitric oxide, thereby gaining mechanistic insight into the significance and timing of oxidative stress during murine infection. We will also test the ability of the B. burgdorferi mutants to infet ticks, to survive the nymphal molt, and to be transmitted back to mice, thereby assessing the contribution of oxidative stress response genes throughout the enzootic cycle. Finally, we will investigate transcriptomic responses to oxidative stress in both wild-type and mutant B. burgdorferi using RNA-seq, allowing us to integrate the genes we have identified within pathways and regulatory networks in the cell. The results of this study will lead us to a better understanding of the basic biology of B. burgdorferi and may also identify new targets for therapeutic interruption of its lifecycle.